Many pathogens utilize clonal variation to diversify their antigenic or virulence factors to promote infection, a process termed virulence modulation (VMO). The aims presented within this application will serve to characterize a novel VMO pathway in Xenorhabdus nematophila that affects virulence towards insects. The regulatory mechanism governing VMO in X. nematophila is not known, however preliminary data suggest a role for the global regulator Lrp in mediating this process. In uropathogenic E. coli, Lrp mediates a clonal variation pathway in a manner that depends on promoter methylation and the activity of accessory proteins. To investigate the role of Lrp in X. nematophila VMO, lrp expression levels will be compared between variants using western blotting and reporter assays. Further experiments will focus on measuring the effects of inducible Lrp expression, promoter methylation, and putative accessory proteins on VMO switching, using the VMO-associated phenotypes as a readout for switch status. Many X. nematophila virulence factors remain to be discovered. Thus, the second aim of this application focuses on the identification of genes in the VMO switch regulon, using ChlP-chip or traditional microarray analyses to identify differentially-regulated genes. A subset of VMO-regulated genes will subsequently be mutated and the resulting strains used to examine pathogenesis in M. sexta in an effort to identify novel virulence factors. Reporters will then be used to study VMO-specific gene regulation during infection. Investigations regarding clonal variation in bacteria to date have mostly focused on those pathways that affect virulence. The third aim of this application serves to address the involvement of the VMO switch in other aspects of the X. nematophila life cycle. Nematode colonization assays will be used to determine whether VMO variants are equally capable of symbiotic colonization within the insect host. In addition, VMO-specific GFP reporters (see above) will be used for in vivo analysis of VMO regulation during nematode colonization and upon entry of colonized nematodes into M. sexta larvae. PUBLIC HEALTH RELEVANCE: These studies serve the broader purpose of establishing a laboratory-tractable, holistic model for investigation of the regulation and overall impact of VMO in host-microbe interactions. In addition, investigation into the diversity of bacterial processes governed by clonal variation will help determine whether such pathways may be good targets for antimicrobial therapy.